. DH DO 

Copy 1 



BACILLUS MORULANS,T$.SP 



A THESIS SUBMITTED IN PARTIAL SATISFACTION OF 

THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

AT THE UNIVERSITY OF CALIFORNIA 



PIERRE AUGUSTE BONCQUET 



December, 1915 



136 



Reprinted from Phytopathology, Vol. VII, No. 4, August 1911 



r. 



BACILLUS MORULANS N. SP. 

A Bacterial Organism Found Associated with Curly Top of the 

Sugar Beet 

P. A. Boncquet 
With Seven Figures in the Text 

The writer, in conjunction with Prof. Ralph E. Smith, has been investi- 
gating a peculiar and fundamentally important plant disease, the so-called 
curly top of the sugar beet and related plants. Two joint publications 1 have 
already been made upon this subject in which there has been briefly an- 
nounced the discovery of a bacterial organism found constantly associ- 
ated with the disease in the tissues of affected plants. It has been stated 
further that, although obtained so regularly and abundantly in cultures 




Fig. 1. Wrinkled Type of Beet Leaves 

Not curly top, but having in the sieve tubes occasional groups of the bacterial 1 
bodies found in great abundance in curly top beets. 

1 Smith, R. E., and Boncquet, P. A. New light on curly top of the sugar beet. 
Phytopath. 5: 103-107. 1915. 

Connection of a bacterial organism with curly leaf of the sugar beet^ 

Phytopath. 5: 335-342. 1915. 



270 



Phytopathology 



[Vol. 7 




Fig. 2. Sugar Beet Plant Typically Affected with Curly Top 



»-3 



1917] Boncquet: Bacillus morulans 271 

from diseased plants or portions of plants, it has not been possible to 
produce curly top by inoculation with cultures of this organism; that the 
same organism has been isolated from the surface of beet seed, the sur- 
face of normal sugar beet leaves and from the soil about the roots of 
sugar beets. Also that certain bodies which seem to represent the same 
organism have been found in great abundance in curly top sugar beets, 
in the interior of the sieve tubes, accompanying a specific lesion in the 
phloem, and that similar bodies, in varying but much less abundance, were 
found in the same tissue in supposedly normal beets or those with various 
morphological irregularities of the foliage (fig. 1). Whatever may be the 
entire significance of the organism in question, its peculiarly abundant 
occurrence in connection with the sugar beet and its apparent relation to 
curly top have seemed to justify its careful study, and it is the purpose of 
the present article to describe more in detail the characteristics of this 
species, to which the name Bacillus morulans has been given. 

THE DISEASE 

The disease of sugar beets called curly top is of annual occurrence 
throughout the sugar beet growing regions of Colorado, Utah, Idaho and 
California. The severity of the disease, however, varies greatly from 
year to year. Some years veritable disasters are produced by curly top, 
thousands of acres of sugar beets being totally destroyed after all the ex- 
pense of preparing the ground and planting the crop has been undergone. 

Symptoms on leaves 

The comparative size of the inner and outer leaves is altered. The in- 
ner leaves are dwarfed, the petiole especially becoming shorter and flatter 
than the normal, while the outer leaves, if already full grown before the 
disease becomes apparent, maintain their natural size and shape and, for 
some time at least, their color, although they may finally turn yellow 
and die prematurely. The first symptom of abnormality plainly visible to 
the eye is a distinct transparency of the finest venations of the youngest 
leaves. This transparency starts at the base of the leaf blade. Gradu- 
ally the abnormality works higher on the leaf until finally the whole leaf 
is affected. The youngest leaves are first to suffer; the older ones (such 
as are not already full grown) show the symptoms as their expansion and 
growth progress. Almost simultaneously with the appearance of the 
transparency of the veins small warty protuberances appear upon the 
veins on the under surface of the leaves, eventually even upon those which 
are of the smallest size visible to the eye (fig. 2) . The margins of the af- 
fected leaves then begin to curl slightly upward so as to expose the lower 



272 Phytopathology [Vol. 7 

surface. As these symptoms gradually increase, working outward from 
the innermost to the outer leaves, black spots soon appear in the principal 
leaf venation and especially in the youngest petioles. These black spots 
often break out through the cortical parenchyma and epidermis, allowing 
a black syrupy substance to ooze out, which upon drying produces in se- 
vere cases a crusty appearance upon the petioles. The interrupted and 
irregular expansion of the leaf venation soon brings about a very char- 
acteristic leaf curl. This curling for the most part is not parallel with 
the main midrib but extends more or less along the margin of the leaf and 
converges at the apex. If the plants are badly affected when very young 
they may be entirely killed; otherwise when the disease is advanced the 
outer leaves become yellow and die while the youngest may remain for 
a long time practically unchanged or occasionally may later resume 




Fig. 3. Sugar Beet Root Tips, Showing Characteristic Bending and Swelling 

growth and become apparently free from the above-mentioned symptoms. 
The latter represents a condition of recovery from the disease. In such 
cases the young leaves are also dwarfed but the venation is normal, the 
warty protuberances are absent and the leaf does not curl. In this condi- 
tion, however, the whole leaf is of a denser green color, harder and thicker. 
The beet seems to have undergone a struggle similar to that which meso- 
phytic plants undergo when they are transplanted to a desert habitat. 
In such cases the roots may develop to normal size even when the tops are 
much suppressed. 

Symptoms on roots 

It is only when the leaves have been thoroughly affected that the roots 
begin to show abnormality. This is characterized by the multiplication 
of rootlets. The older ones begin to die while at their base new ones push 
forth. This continuous destruction of the rootlets brings about a warty 
appearance, the bases of the masses of rootlets protruding slightly from 
the surface of the beet. On careful examination it will be observed that 
each rootlet, after it reaches an appreciable length and before it dies, has 



1917] Boncquet: Bacillus morulans 273 

several abnormal bendings; the angle of each bend is slightly swollen and 
if the rootlet is far advanced the swollen region appears to be necrotic 
(fig. 3). When the main root is cut transversely the successive rings of 
vascular tissue appear discolored. On careful examination it will be 
observed that the phloem is the only part of the vascular system which 
suffers severely. This phloem discoloration is observed more or less 
throughout the whole system in the veins as well as in the roots. This, 
however, only becomes apparent to the naked eye when the disease has 
reached its severest aspect. 

Cause of the disease 

It was E. D. Ball 2 who discovered that the sting of the insect Eutettix 
tenella Baker is a necessary factor in the causation of this disease. His 
observations were confirmed by Shaw, 3 and very fully tested and confirmed 
by Smith and Boncquet. The latter, however, together with Hartung, 4 
proved a fact which had previously been suspected, that Eutettix tenella 
is not the fundamental factor in the causation of this disease, but rather 
must be a carrier of a second factor, presumably a parasitic micro-organ- 
ism. This discovery made very important a thorough search for and study 
of all micro-organisms which possibly could be found in connection with 
the disease, and it is with this portion of the study of curly leaf that the 
present article has to do. 

BACTERIOLOGICAL INVESTIGATIONS 

The methods and detailed results through which the conclusion was 
reached by the writer that Bacillus morulans inhabits constantly and 
specifically plants affected with curly top, as well as occurring in certain 
other situations may first be described. It was decided at the outset to 
make a very thorough and accurate search for any organism which might 
be present in the tissues of plants affected with the disease. The unfav- 
orable or at best uncertain results reported by previous investigators 
along this line led to the belief that the problem would be a difficult one, 

2 Ball, E. D. The beet leaf hopper. Utah Agr. Exp. Sta. Ann. Rept. 16: 16. 
1905. 

— • The Genus Eutettix. Proc. Davenport Acad. Sci., 12: 41 and 84. 1907. 

— -■■■ - The leaf hoppers of the sugar beet and their relation to the "curly leaf" 
conditions. U. S. Dept. Agr. Bur. Ent. Bui. 66, pt. 4. 1909. 

3 Shaw, H. B. The curly top of beets. U. S. Dept. Agr. Bur. PI. Ind. Bui. 181. 
1910. 

4 Boncquet, P. A. and Hartung, W. J. The comparative effect upon sugar beets 
of Eutettix tenella Baker from wild plants and from curly leaf beets. Phytopath. 
5: 348-349. 1915. 



274 Phytopathology [Vol. 7 

but at the same time a study of the nature of the disease had led to a very 
strong feeling that some parasitic micro-organism, of which the insect 
Eutettix tenella was presumably a carrier or secondary host, must be in- 
volved in this disturbance. Assuming then that the sought-for organism 
would be an obscure one and difficult to demonstrate by ordinary cultural 
or histological methods, various special and somewhat elaborate culture 
methods were attempted. 

Preparation of media 

The following media which seemed most promising fortius purpose were 
prepared : 

Filtered beet juice. For this purpose the plants from which the juice was desired 
were washed as thoroughly as possible in sterilized water and then ground fine in a 
meat grinder. In most cases 100 cc. of distilled water was added to each 100 grams 
of beet pulp and the mass was then covered and left standing for two hours. The 
crushed material was subsequently put into clean cheesecloth and the juice squeezed 
out in a press. The juice thus obtained was subsequently diluted twice its volume 
with n/6 salt solution. Various degrees of dilution have been employed, however, 
from the original juice up to about ten to one, either in salt solution or water. In 
some cases the solution was then titrated and brought to the neutral point of phenol- 
phthalein with sodium hydroxid. This juice was now clear, slightly brown and 
passed easily through a common filter paper. After it had been filtered through 
paper it was passed through a medium-dense Berkefeld filter candle for purposes of 
sterilization. An apparatus was especially devised for this purpose, a form of which 
is described in another article in the present number of Phytopathology. In order 
to be sure that no contamination had occurred during the manipulation, the tubes 
after filling were kept in the incubator for two days at 30°C. It is believed that this 
apparatus and method is worthy of considerable employment in the preparation of 
culture media for use in plant pathology. 

Aseptic, unheatedbeet slices. These were prepared in the following manner: Sound, 
healthy beets were selected, thoroughly cleaned and immersed in boiling water for 
three minutes, in this way sterilizing the surface but not heating or changing in any 
manner the tissues deeper in the beet. They were then cut into slices with a care- 
fully sterilized knife. Each slice was then put into a sterile petri dish into which 
previously ordinary nutrient agar had been poured. 

Beet broth. Three hundred grams of beet leaves were cut into small pieces and 
boiled for an hour in 0.5 litre of water. Water was then added to make up to 1 litre 
and left standing for two hours. It was then filtered through cotton and 500 cc. of 
this beet extract added to 1 litre of Liebig's broth. The Liebig's broth had previ- 
ously been prepared in the following way: 2 grams of Liebig's extract, 10 grams of 
Witte's peptone, and 5 grams of sodium chlorid were added to 1 litre of water. This 
medium was subsequenty neutralized to phenolphthalein with sodium hydroxid 
and after addition of the beet juice was brought up to 0.5 per cent acidity with 
malic acid. The same medium was also prepared with an increased proportion of 
beet extract. 



1917] Boncquet: Bacillus morulans 275 

Artificial media. A protein- and peptone-free medium was composed with the 
supposition that the organism did not attack the higher nitrogen compounds of the 
beet. Therefore several of the amino acids were used as the nitrogen supply. Ala- 
nin, leucin and tyrosin were used. Asparagin, although not an amino acid was also 
considered a possible favorable source of nitrogen for the parasitic organism. All 
these compounds were used in a dilution of 0.5 gram to 1 litre of water. The neces- 
sary minerals were added in the following form and proportion: 

Magnesium sulfate 0.2 gram 

Ammonium phosphate 0.5 gram 

Potassium nitrate 0.2 gram 

Calcium hydroxid 5 cc. of a saturated solution 

Ferric chlorid trace 

These artificial media were sterilized in the Arnold sterilizer for fifteen minutes 
upon three consecutive days. Special glycerin and glucose media were also pre- 
pared. For this purpose 1 per cent glycerin was added to a part of the asparagin 
medium. So also 5 per cent glucose was added to another portion. The glucose 
medium was especially used for anaerobic purposes. 

Other media. Ordinary media such as nutrient bouillon, potato glucose bouillon, 
bean pods, milk, litmus whey, nutrient agar and nutrient gelatin were prepared ac- 
cording to the standard methods. 

Methods attempted for separating parasites from the plant 

In order to separate the assumed parasites from the plant and obtain 
them in pure culture the following technique was used : 

Surface-disinfected plant parts placed in culture medium. The usual 
method employed in this sort of work consists in soaking the tissue to be 
employed for a given length of time in mercuric chlorid and then washing 
off the same with sterilized water before placing the tissue in the culture 
medium. A need of standardizing this method was felt, inasmuch as 
there is no assurance, as it is usually described, whether on the one hand 
the disinfection was sufficiently thorough to kill all surface organisms or 
whether on the other hand the material was washed sufficiently to remove 
all the mercury and prevent its being carried over into the culture medium. 
The method consists in dry sterilizing a number of cotton-plugged flasks 
of 50 cc. capacity or any other desired size. At the same time larger 
flasks, likewise cotton-plugged and filled with distilled water, are made 
sterile in the autoclave. Other requisites are supplies of 95 per cent 
alcohol and 1 to 1000 solution of mercuric chlorid in water. The mate- 
rial from which cultures are desired, after thorough wiping with cotton 
swabs in 95 per cent or absolute alcohol in a photographic tray, is cut 
into convenient sized fragments, but no smaller than necessary. These 
are placed in one of the empty sterilized flasks and covered for a moment 
with the alcohol for the purpose of removing air bubbles. The alcohol is 
immediately poured off again and the flask nearly filled with mercury 



276 Phytopathology [Vol. 7 

solution so that all the material will be submerged. This is allowed to 
remain for the desired length of time, depending upon the nature of the 
tissue. The petioles and main veins of sugar beet leaves, especially fairly 
old leaves, will usually stand twenty minutes, but with leaf blades and 
other more delicate material ten minutes has been found the maximum 
time which can be used without too severe burning. Cut surfaces will 
naturally absorb more of the solution than those protected by the natural 
covering of the plant, and this can be taken into account both in consid- 
eration of the length of time which the tissue will stand without being 
burned by the mercury and also the time necessary for washing it out 
again. On this account it is best to cut the tissues as little as possible 
before disinfecting. After the desired time has elapsed a piece of brass 
wire gauze, bent to form a cap over the mouth of the flask, is sterilized in 
the flame, placed in position and the mercury poured off. The flask is 
then filled with sterilized water from the large flask and the water of this 
first washing, after having the material well shaken up in it, is poured off 
immediately and more water poured in. The process of pouring off and 
refilling is then continued at gradually increased intervals; the length of 
each must depend upon the nature of the material. It was found, how- 
ever, that if the amount of material in the flask is comparatively small in 
proportion to its capacity, which should always be the case, six changes 
of water, extending over a period of two hours, is amply sufficient in every 
case. In this case the first five changes can be made during the first hour 
and the last one at the end of the second horn*. If one wishes to practice 
extreme caution the mouth of the flask may be flamed and the cotton 
stopper replaced after each change of water, but this has not been found 
necessary so long as the amount of water is sufficient to thoroughly sub- 
merge all the material. In our work the wire screen is usually left over 
the mouth of the flask and this is freshly flamed before each change of 
water. After the process is completed the material is taken out of the 
last water with flamed forceps, broken into small pieces if necessary and 
thoroughly crushed with the same instruments and dropped into the cul- 
ture liquid. 

Piece cultures. It was thought that a gradual adaptation from the 
plant in which the organism is living to the medium in which it was at- 
tempted to grow it might be necessary to insure success; therefore the dis- 
eased tissue was so transferred as to disturb as little as possible the cells 
of the beet. For this purpose glass tubes were drawn out to 2 mm. diame- 
ter. After sterilizing by heat they were aseptically inserted into the dis- 
eased regions of the beet to a depth of 1 cm. The tube was then with- 
drawn, bringing with it a portion of the beet tissue and the terminal 
part containing the tissue was carefully broken off with sterilized forceps 



1917] Boncquet: Bacillus morulans 277 

and dropped into the medium. In this way both ends of the tissue slightly 
protruded from the glass tube and came into direct contact with the cul- 
ture medium. The slow diffusion of the latter was supposed to secure a 
gradual change of habitat in such a way as not to hinder too severely the 
growth of the parasite. Tissue was thus removed from the petiole, from 
inside the root and from the larger veins of the leaf, after surface sterili- 
zation with a flame or boiling water, afterwards cutting into the interior 
with a sterilized knife and then introducing the glass tube to take out a 
small core of tissue. 

Results of isolation experiments 

The various special methods described were carried out very carefully. 
The result was that in almost every instance cultures from curly top tis- 
sue in all the various media described, and especially those which con- 
tained glucose, showed a heavy growth after twelve hours of incubation 
at 20°C. This result was practically uniform wherever diseased material 
had been used. Occasionally growth also appeared in cultures from sup- 
posedly normal plants, but in by far the great majority of cases such cul- 
tures remained sterile. This seemed to indicate that the organism was 
not peculiarly difficult to isolate, judging from its abundant growth on such 
a wide variety of media. Nevertheless a painstaking work was under- 
taken in order to complete the thorough study which had been planned. 
The anaerobic cultures also proved to be invaded by the same organism. 
Here, however, the growth was less abundant and extremely slow. Sev- 
eral days elapsed before any colonies were visible. A great deal of effort 
was further spent on work with all kinds of media but always the same 
organism grew abundantly. Contaminations naturally occurred now and 
then but the fact was most decidedly apparent that the one species an- 
nounced by Smith and Boncquet predominated in the tissues of curly top 
plants to the practical exclusion of all others. The special culture meth- 
ods described above are given in some detail, inasmuch as they may con- 
tain suggestions of value in similar work. Having found that this organ- 
ism grew so easily and abundantly upon ordinary media, the use of special 
preparations was abandoned in the attempts to isolate the organism from 
plant tissues and the work was carried on entirely with standard bouillon 
to which 5 per cent glucose had been added. The object of the glucose was 
to promote the growth of the characteristic zoogloeae of this organism, 
rendering its identification in the original tubes easy without plating. By 
occasional plating, as a check on the work, it was soon possible to identify 
this organism very accurately by microscopic examination of tubes which 
showed the characteristic ring formation at the surface of the liquid. The 



278 



Phytopathology 



[Vol. 7 



TABLE 1 

Results of bacterial isolation experiments by cultural methods 
(Tubes incubated at 28°C. Disease means curly top) 



MATERIAL 


NUMBER OP 
TUBES 


RESULT 


Petioles of curly top beets 


6 


5 tubes developed B. morulans 
within two days 


Petioles. of normal beets 


6 


Tubes remained clear for a week, 
when they were discarded 


Petioles of diseased beets 


9 


8 tubes developed morulans 


A leaf showing curly top symp- 


5 


All developed morulans 


toms on half of blade and in cor- 






responding half of petiole. 






Other side appeared normal. 






This material from affected half 






of petiole with black streaks 






Corresponding half of blade 


3 


2 tubes developed morulans 


Normal-appearing half of petiole 


6 


5 tubes developed morulans two 
days later than those from 
blackened part 


Normal-appearing side of blade 


3 


No development 


Petiole of a diseased center leaf 


4 


3 with morulans, 1 doubtful 


Petioles of good-sized leaves from 


10 


2 tubes from 1 leaf both with moru- 


5 different normal-appearing 




lans; others all clear 


beets 






Typically diseased leaf with very 


6 


All developed morulans 


slight dark streaks in the petiole 






Badly affected petiole of same 


4 


All developed morulans 


beet. Pieces cut out with 






flamed scalpel 






Petioles of 4 beets from insect- 


8 


Tubes from 2 plants remained 


proof cage. No sign of disease 




clear; those from other 2 became 
slightly cloudy after several 
days, but no morulans 


Petioles of 2 slightly diseased 


13 


All tubes apparently containing 


leaves. Pieces cut out with 




pure cultures of morulans 


flamed scalpel 






A yellowish aster leaf 


2 


Remained clear 


An old yellowish beet leaf without 


4 


Some fungous growth. No moru- 


curly top. Tissue still sound 




lans 


Leaves of a somewhat abnormal- 


6 


Some growth, but no morulans 


appearing beet but not with 






curly top 






Healthy-appearing leaves of aster, 


24 


11 tubes with fungi and bacteria, 


chrysanthemum, dahlia, toma- • 




remainder clear. No morulans 


to, bean, lettuce and raddish 




found 


Petiole of typically diseased leaf, 


6 


Very abundant growth of morulans 


no disinfection 




intermixed with other organisms 



1917] 



Boncquet: Bacillus morulans 



279 



TABLE 1— Continued 



MATERIAL 


NUMBER OF 
TUBES 


RESULT 


Petiole of slightly diseased leaf 


2 


Both very abundant morulans 


Blade of same between veins 


2 


Both remained clear 


Typically diseased leaf; scraped 


4 


3 tubes developed morulans. 1 


out interior portions of petiole 




doubtful 


with flamed scalpel after clean- 






ing off epidermis 






A young leaf visibly affected on 


4 


All developed morulans 


one side and very slightly at 






the base of the other side. Tis- 






sue taken from the most dis- 






eased side at base 






Terminal portion of diseased side, 


4 


2 developed morulans; 2 clear 


less visibly affected 






Slightly affected base of other side 


4 


1 developed morulans; 3 clear 


of same leaf 






Not visibly affected terminal por- 


4 


All remained clear 


tion of last 







Note. — The last four are from the leaf illustrated in Phytopathology 5: 106. 
The most elaborate precautions were taken to secure perfect surface disinfection and 
avoid contamination. These tubes in which growth appeared were plated out and 
found to contain pure cultures of morulans. The leaf was perfectly sound, showing 
only a slight roughening of the veins on the affected portion. 

appearance to the eye of this ring, supplemented by microscopic examina- 
tion, finding it to be composed of the characteristic zoogloeae, supple- 
mented by occasional plating, is amply sufficient to identify this organ- 
ism. A number of typical examples of isolation experiments with sugar 
beets are shown in table 1. 

Several hundred illustrations similar to those shown in table 1 might 
be given. The results varied somewhat with the perfection of technique 

TABLE 2 
Bacillus morulans upon sugar beet seed 



MATERIAL 


NUMBER OF 
TUBES 


RESULT 


Beet seed imported from Ger- 


10 


At least 7 developed an abundance 


many, 1 dropped into each bouil- 




of morulans, mixed with other 


lon tube, with no previous treat- 




organisms 


ment 






Similar seed previously soaked for 


10 


All clear 


twenty minutes in mercuric 






chlorid and washed in sterilized 






water 







280 



Phytopathology 



[Vol. 7 



and in individual cases, but a mass of evidence was collected to indicate 
that this organism exists regularly in the interior of the foliage of sugar 
beets where the visible symptoms of curly top occur and that it does not 
develop in cultures from normal foliage or even the normal-appearing por- 
tions of partially affected leaves; also that it does not occur in the interior 
of beet leaves which may be yellow or sickly from ordinary causes. 

Cultures from seed. Many attempts similar to those shown in table 
2 were made to isolate the organism from sugar beet seed. The uniform 
result was that almost every unsterilized beet seed dropped into a tube 
of bouillon developed a very luxuriant growth of Bacillus morulans. 

TABLE 3 

Bacillus morulans from soil 



MATERIAL 


NUMBER OF 
TUBES 


RESULT 


Pinches of soil from about the 
roots of a diseased beet 

Pinches of soil from about the 
roots of a normal beet in insect- 
proof cage 


4 

4 


Morulans was abundant in several 

of the tubes 
Some morulans present in the 

mixed growth resulting 



Cultures from soil. That the organism is present in some soils is indicated 
by the data presented in table 3. The work was rather crude but cer- 
tainly B. morulans was abundant in the soils examined. 

Cultures from unsterilized foliage. Cultures made from unsterilized 
leaves of the sugar beet (table 4) show that the organism is common as a 
saprophyte upon the leaves of the plant, but in all cases when leaves simi- 
lar to these were thoroughly disinfected no growth was obtained. 

TABLE 4 
Cultures from unsterilized foliage 



MATERIAL 


NUMBER OF 
TUBES 


RESULT 


Leaves of normal beets 


10 


Several contained an abundance 
of morulans 



Unsterilized leaves of many other plants were also tried but the resulting 
growth was so mixed that no safe conclusions could be drawn. The only 
certain developments of morulans occurred in tubes inoculated with pieces 
of chrysanthemum leaves. 

Cultures from sugar beet leaves with types of disease other than curly top. 
The fact that bodies resembling bacteria have been seen with the micro- 
scope in sugar beet leaves not affected with curly top, but affected with 



1917] 



Boncquet: Bacillus morulans 



281 



various types of morphological abnormality, has been referred to in our 
recent article in Phytopathology. These various foliage types have never 
been definitely classified or described nor has the extent of the occurrence 
of this organism in other tissues been learned. In this cultural study, 




Fig. 4. Mottled Leaf of Sugar Beet 



however, two at least of these forms gave the organism in abundance (table 
5). In one of these, which we shall call mottled leaf (fig. 4), the blades 
of the leaves are strongly mottled with green and pale areas in a very 
characteristic manner. In the other type which we call black edge or 
black tip (fig, 5), the green leaves begin to die and turn decidedly black 



282 



Phytopathology 



[Vol. 7 



about the margin or at the tips, with a yellow band of tissue between the 
black and the normal green. In some such leaves the development is 
very much suppressed, even to a point where the leaf consists of scarcely 
more than a petiole with a small blackened tip. 






Fig. 5. Black Edge and Black Tip of Sugar Beet Leaves 
Black tip (left) and black edge of sugar beet leaves 

Relation of mottled leaf and black edge to curly top. For some time it 
was believed that these conditions represented types or stages of curly 
top. Later, however, it was found that they occurred under circumstances 
which made this seem practically impossible. Further, when the experi- 
ment was tried of placing Eutettix tenella upon such plants the typical 
disease developed promptly. 



1917] 



Boncquet: Bacillus morulans 



283 



TABLE 5 

Cultures from sugar beet leaves with types of disease other than curly top 





NUMBER OF 




MATERIAL 


TUBES 


RESULT 


Yellowish area of young "black 


2 


Both gave a strong growth of 


edge" leaf taken from between 




morulans 


black and green portions. 






Thoroughly disinfected 






Similar to last, but not disinfected 


4 


Very vigorous and apparently 
nearly pure growth of ynorulans 


Similar material, disinfected 


4 


All with morulans 


Similar material, disinfected 


4 


All with morulans 


Typical "mottled leaf;" not dis- 


9 


All produced morulans in abun- 


infected. Blade, petiole and 




dance 


veins 






Petiole of "black edge" leaf, dis- 


4 


All appear to have pure cultures 


infected 




of morulans 


Petioles of normal appearing leaves 


5 


All tubes clear 


from healthy plant. Very 






carefully disinfected 






Petiole of decidedly "mottled 


4 


Very vigorous growth of morulans 


leaf." Inner tissue removed 






with flamed scalpel 







Further study of the organism, which was uniformly present in diseased beets 

Although the organism was able to grow most abundantly on the com- 
mon culture media, the peculiarity of this growth under all circumstances 
was of such a nature that for some time a continual contamination was 
suspected. Each separate colony seemed always to have two kinds of 
bacteria, very distinct in form. Very active bacteria were always observed 
at the edge of the colonies, while capsultated bacilli were generally ob- 
served in the middle. Therefore, before any further study of the organ- 
ism was taken up, repeated efforts were made to separate these two widely 
distinct forms. For this purpose, the calcium carbonate and the India 
ink method for separating the individual organisms previous to plating 
them were resorted to. 

Calcium carbonate. To 10 grams of calcium carbonate enough water 
was added to form a milky paste. This was subsequently introduced into 
a 200 cc. Erlenmeyer flask and sterilized in the autoclave. After the nec- 
essary cooling several young colonies of the bacteria were introduced into 
the semi-liquid mass and shaken for two hours so as to separate each indi- 
vidual organism from the other. From this paste, several plates were 
poured in the usual manner. They were incubated at 37°C. and closely 
examined as soon as any sign of development occurred. 



284 Phytopathology [Vol. 7 

India ink method. For this purpose special Chinese ink, prepared by 
Griibler, (Punkttusche) was used. A 15 per cent nutrient gelatin was 
made and poured into clay-covered petri dishes. Special care was given 
to prevent condensation water from flooding the medium. The Griibler's 
ink was diluted twenty times with n/6 glucose solution and sterilized in 
the autoclave. In a sterilized, empty petri dish ten drops of the ink were 
put in a row. The first drop was inoculated with a small amount of bac- 
teria from a twelve-hours-old streak culture. The bacteria were thor- 
oughly mixed with the ink of the first drop. Then a loop of this was trans- 
ferred to the second drop and also thoroughly mixed. This transfer was 
repeated in the same way with the remaining drops in the dish. From 
the tenth drop, with a sterilized drawing pen, a small amount was taken. 
Small dots were made with the pen on a gelatin plate in such a way that 
the surface pellicule of gelatin remained uninjured. These ink dots were 
left to dry for two minutes then covered with a sterilized cover-glass. A 
small drop of immersion oil was subsequently applied to the cover-glass 
and the whole petri dish was brought to the microscope for examination. 
Each 'black point was then examined with microscope until one was 
found which contained one single organism. The organism appeared as a 
translucent dot on a black field. Its development was closely followed; 
the first division was distinctly noticed after half an hour; it multiplied 
rapidly; all the individuals were motile; they liquefied the gelatin slightly 
and moved about very briskly in the liquid under the cover-glass. After 
six hours some of the organisms became sluggish and gradually lost their 
motion. They increased in size and formed a capsule. Repeatedly they 
divided in the same capsule, stretching the jelly-like membrane more and 
more. The newly formed organisms within the original capsule also en- 
capsulated in their turn (figs. 6 and 7). At the same time the individuals 
on the rim of the colony multiplied and remained motile. The double 
form of the bacillus was in this way clearly explained and proved. 

IDENTITY OF THE ORGANISM FOUND IN CURLY TOP BEETS 

A study of the literature of the subject shows that the greatest similarity 
to our organism of any described species is presented by that described by 
Arthur and Bolley 5 as Bacterium Dianthi as the cause of a leaf spot of the 
carnation. In its morphology, so far as described by these writers, this 
organism is very similar to ours, the resemblance being made pronounced 
by the development of characteristic zoogloeae. In biological behavior, 
however, the two organisms cannot be accurately compared, since the 

5 Arthur, J. C. and Bolley, H. L. Bacteriosis of carnations. Indiana Agr. Exp. 
Sta. Bui. 59. 1896. 



1917] Boncqtjet: Bacillus morulans 285 

work of Arthur and Bolley was carried on at a time when bacteriological 
technique was not standardized upon modern lines. One noticeable dif- 
ference exists in respect to growth upon an acid medium, B. Dianthi being 
said to grow best under such circumstances, which is not the case with 
our organism. The description of the bacterial organism given by Arthur 
and Golden 6 and again by Miss Cunningham 7 as the cause of the so-called 
Indiana sugar beet disease, is similarly subject to uncertainty, but if this 
work was accurately done the organism must certainly have been differ- 
ent from ours in that it is said to be a particularly active gas former, which 
feature is totally lacking in our organism. It seems proper to mention 
here, however, the fact that Professor Arthur in a recent personal letter 
states that the accuracy of all this early work performed under his direc- 
tion is open to some doubt on account of the undeveloped condition of 
bacteriological technique at the time and he expresses the opinion that 
the organisms found by Bolley, Miss Golden and Miss Cunningham were 
very likely identical. 

The organism described in the unpublished work of Schneider 8 as Bacil- 
lus calif orniensis, which was isolated from curly top beets in California, 
seems again in its morphological characteristics to be entirely similar to 
ours and we feel little doubt that Schneider and the present writer had 
the same organism before them. Schneider found his organism also very 
abundant in sugar beet soils and upon the surface of the plants. He at- 
tributes a stimulative effect to this species, when applied in pure culture 
to sterilized beet seed or to the foliage of young plants. 

The organism described by Diiggeli 9 as being abundantly and often ex- 
clusively present upon the surface of various plants and seeds, seems also 
very similar to ours in form and size, formation of zoogloeae, color, sapro- 
phytic habitat and most biological characters. This was named by Diig- 
geli Bacterium herbicola aureum, but "said to be the same as the Bacillus 
mesentericus aureus, isolated by Winkler from the surface of plum leaves." 
The latter statement confuses the identification. 

DESCRIPTION OF THE ORGANISM 

Summing up the whole situation, we feel justified in describing our 
organism as a new species on account of the incomplete and doubtfully 

6 Arthur, J. C. and Golden, K. E. Disease of the sugar beet root. Indiana Agr. 
Exp. Sta. Bui. 39, pt. 3:54. 1892. 

7 Cunningham, C. A. A bacterial disease of the sugar beet. Bot. Gaz. 28: 177— 
192. 1899. 

8 Schneider, A. The California beet blight. Spreckels Sugar Co. Exp. Sta. 
Rept. 23: — . 1906. (Unpublished.) 

9 Centbl. Bakt. II, 12: 602 and 695; 13: 56 and 198. 1904. 



286 Phytopathology [Vol. 7 

accurate descriptions of those species which more or less resemble it, and 
the fact that none of them corresponds throughout. 

Bacillus morulans n. sp. 

Morphology 

Vegetative cells. Grown in Liebig bouillon for sixteen hours at about 
20°C, oval to short rods, single or in pairs. Grown at 37°, short rods in 
pairs or in short chains. 

Size. Length 1.5 n; breadth 0.9 n; extreme length from 1.5 to 2 n. 

Capsules. Easily observed in 1/1000 glycerin bouillon after twenty- 
four hours and also in milk media (figs. 6 and 7). 

Motility. Very active on agar and in bouillon, when grown at 37° for 
twelve hours. 

Flagella. Stained by Zettnow's method; four long peritrichial flagella 
(figs. 6 and 7). 

Pleomorphism. Cocciforms observed in glucose bouillon tubes and 
blood serum media after thirty days. 

Stain. Easily with watery fuchsin, decolorized by Gram's method. 

Cultural features 

Gelatin plate neutral to phenolphihalein. Form, round to irregular; 
surface elevation, flat to convex contoured; internal structure, refraction 
strong, hyaline, moruloid; zoogloeae very marked; edges, entire to undu- 
late; optical characteristics transparent to butyrous; consistency, viscous. 
Each colony is surrounded with many secondary colonies, appearing as 
small, oily drops of high refractive power. The appearance of zoogloeae 
is very noticeable in the middle of the colonies. 

Gelatin plates 1.5 acid to phenolphihalein. The entire mass is a zoogloea, 
lobed and irregular in outline; the colony is slightly colored; orange-yel- 
low, no surrounding colonies noticed. 

Gelatin streak. After five days: growth, linear; margin, continuous; 
surface relief, flat to convex; light transmission, butyrous; color, yellow- 
orange; luster, glistening; consistency, viscous. The water of condensa- 
tion has a yellow sediment. 

Gelatin stab. After twenty-two hours, top growth: size, 5 mm., irregular 
contoured; pulvinate to capitate, light orange in color; viscous in consist- 
ency, luster shining. 

Gelatin streak. Filiform to slightly beaded. After fifteen days, lique- 
faction of medium; crateriform with a yellow sediment. After twenty 
days, liquefaction stratiform; yellow pigment, decreased by absence of 
oxygen. 



1917] 



Boncquet: Bacillus morulans 



287 




Fig. G. Morphology of Bacillus morulans 

1, A chain of two individuals, showing flagella. 

2, 3, 5, development of capsule and zoogloea from one individual. 4> mass of 
zoogloeae. 




Fig. 7. Bacillus morulans, Showing Capsules and Flagella 



288 Phytopathology [Vol. 7 

Agar. The colonies are extremely variable according to the density of 
growth, the moisture and the temperature. 

Milk. Peptonisation of casein in fifteen clays at 37°. The reaction is 
alkaline to Azolitmin. 

Litmus whey. Remains clear, alkaline reaction. 

Bouillon tubes. Opacity begins after eight hours at 37°, a pellicule 
forms in twenty-four hours or less. The color of the pellicule and the 
ring is dull, soft gray ; thick, viscous and consists of conglomerate zoogloeae. 
These are generally oval, but may be linear and all united in chains. 

Deposit, forms after two days incubation at 37°. Deposit is in the 
beginning slight, and finally yellow. The amount of deposit and the in- 
tensity of the color increases, however, rapidly. After ten clays, a de- 
cidedly deep yellow-orange has developed. The deposit is compact and 
viscid on agitation. 

Potato streak. After twenty four hours at 37°. Size, 2 mm.; sharp, 
linear; margin, continuous; color, yellow, homochromous ; luster, glisten- 
ing; texture, homogeneous. No liquefaction of potato and no gas 
formation. 

Physical and biochemical features 

Reaction. In carbohydrate-free media the reaction is alkaline; in carbo- 
hydrate media, the reaction is acid, except in lactose, where the reaction 
is slightly alkaline. See table 6. 

Nitrate Liebig broth. After twenty-four hours at 37°, strongly reduced 
to nitrite. 

Indol. Not produced in peptone solution after ten clays. 

Optimum temperature. 37°, measured by the amount of acid produced 
in 1 per cent glucose after five clays. Acidity was 2.5. 

Thermal deathpoint. Six-hours culture in bouillon; 54°C. in ten minutes. 

Carbohydrate fermentation. Shown in table 6. 

Resistance to mercuric chlorid. Six-hours culture on bouillon agar streak 
killed in 1/25,000 to 1/30,000 in ten minutes. 

Relative growth in acid and alkali n media. Determined by the appear- 
ance of cloudiness in the tubes. Grows best on neutral or slightly alkalin 
media. Five per cent in acid apparently stops all growth; 7 per cent in 
alkaline: same. 

Gas production. No gas is produced. See table 6. 

Relation to free oxygen. Aerobic; facultative anaerobic. 



1917] 



Boncquet: Bacillus morulans 



289 



TABLE 6 

Carbohydrate fermentation of Bacillus morulans 

(Incubation: 37°C, medium neutral to azolitmin) 



medium: 1 per cent 




« 
H 
H 
fa 

< 


00 

< 

« 

K 
a 
n 
fa 


>> 
< 
a 

« 

K 

fa 
■< 


< 



« 

H 

fa 

■< 


m 

p 

« 

a 

H 

fa 

< 


a 

o H 


PS 

a 


g 

s> 

o 

Oh 

H 

a 


a 
1 
fa 




5 


REACTION 


PER CENT 
CARBOHYDRATE 


Closed 
arm 


Open 
arm 














xio-* 














Dextrin 








AB* 


AB* 


AB* 







P 


a 


a 


acid 


alk. 


Inulin 






























a 
a 


a 
a 


X 

a 









Amygdalin 





Salicin 


A 


A 


A 


A 


A 


1.8 





P 


a 


a 


acid 


acid 


Glycerin 











A 


A 


0.1 





a 


a 


b 


acid 


acid 


Lactose 



A 



A 


B 
A 


B 
A 


B 
A 


0.2 
2.3 






a 
b 


a 
b 


a 
P 


basic 
acid 


basic 


Laevulose 


acid 


Galactose 


A 


A 


A 


A 


A 


2.1 





b 


b 


P 


acid 


acid 


Dextrose 


A 


A 


A 


A 


A 


2.5 





b 


b 


b 


acid 


acid 


Saccharose 


A 


A 


A 


A 


A 


1.6 





P 


b 


P 


acid 


acid 


Mannit 


A 


A 


A 


A 


A 


1.3 





P 


a 


X 


acid 


acid 


Maltose 


A 


A 


A 


A 


A 


1.3 





P 


a 


P 


acid 


acid 


Rhamnose 


A 


A 


A 


A 


A 


1.3 





P 


a 


X 


acid 


acid 



Note. — A, acid; B, basic; p, permanent; b, abundantly present; a, absent; x, 
more or less present; *, acid on top and basic in tube. 

Pathogenesis 

One loop from a twelve-hours-old streak culture on bouillon agar intro- 
duced intravenously in a rabbit, caused death within twenty-four hours. 

On Dianthus incarnata. The young unfolded leaves, when unrolled and 
covered with an abundant suspension of bacteria, developed small necrotic 
regions. The necrotic regions are watery and translucent on the edges, 
slightly elongate or irregular in outline, following the venation. The in- 
side of the necrotic regions is slightly brown. 

University of California 
Berkeley, California 



f 



